Utilizing the innate immune system to deliver therapeutic agents

ABSTRACT

In one aspect, the invention provides a composition comprising at least one monocyte comprising an agent that increases monocyte homing to a site of injury, and an effective amount of a drug. In another aspect, the invention provides a method of using the composition to deliver a drug to a site of injury.

CROSS REFERENCE TO RELATED APPLICATIONS

The application claims priority to U.S. Provisional Patent ApplicationNo. 62/512,284, filed May 30, 2017, which is incorporated herein byreference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under Contract No. R01HL130037 awarded by the National Heart, Lung, and Blood Institute(NHLBI) of the National Institutes of Health (NIH). The government hascertain rights in the invention.

BACKGROUND OF THE INVENTION

Efficient drug delivery to a specific site of injury within a region ofthe body of the patient in order to minimize off-target effects is acontinuing challenge in the pharmaceutical arts. This is especiallychallenging in regions of the body, such as the brain, to which accessis highly regulated by endogenous systems. There is a need in the artfor compositions and methods that facilitate the delivery of therapeuticagents to difficult-to-target sites of injury.

SUMMARY OF THE INVENTION

In one aspect, the invention provides a composition comprising at leastone monocyte comprising an agent that increases monocyte homing to asite of injury, and an effective amount of a drug.

In another aspect, the invention provides a method of delivering a drugto a site of injury of a patient comprising administering to the patienta composition comprising at least one monocyte comprising an agent thatincreases monocyte homing, and an effective amount of a drug, whereinthe at least one monocyte travels to the site of injury where the drugis released from the intracellular space, thereby delivering theeffective amount of the drug to the site of injury.

In various embodiments, the agent that increases monocyte homing is aCCR2 modulator.

In various embodiments, the agent that increases monocyte homing isdexamethasone.

In various embodiments, one or more of the agent and the drug arelocalized within one or more particles within the monocyte. In variousembodiments, the particle has a cross-sectional dimension between 1 nmand 50 Mm.

In various embodiments, the agent and the drug are both co-localizedwithin one or more particles within the monocyte. In variousembodiments, the particle has a cross-sectional dimension between 1 nmto 50 μm.

In various embodiments, the drug is a drug that affects monocyte ormacrophage behavior and may be the same or different than the agent thatincreases monocyte homing to a site of injury. In various embodiments,the drug that affects monocyte or macrophage behavior is interleukin-4,interleukin-10, interferon-γ or dexamethasone. In various embodiments,the drug is a drug that affects liver function or health. In variousembodiments, the particle comprises a cationic polymer.

In various embodiments, the particle comprises a targeting ligand. Invarious embodiments, the targeting ligand promotes selective uptake byan endogenous monocyte.

In various embodiments, the particle comprises a molecule that enhancesendosomal escape.

In various embodiments, the particle comprises a fluorescent dye. Invarious embodiments, the fluorescent dye is Nile red or Cy5.

In various embodiments, wherein the particle comprises a hydrophobicpolymer core. In various embodiments, the hydrophobic polymer corecomprises a poly(lactic-co-glycolic acid).

In various embodiments, the particle further comprises a coating.

In various embodiments, the particle is a phospholipid vesicle and thephospholipid vesicle comprises one or more concentric lipid layers.

In various embodiments, the drug is released from the intracellularspace by monocyte-to-macrophage differentiation or an injury-specificbiological cue.

In various embodiments, the drug is a prodrug that masks a functionalagent and the functional agent is released at the site of injury.

In various embodiments, the site of injury is in the brain of thepatient.

In various embodiments, the injury is a traumatic brain injury.

In various embodiments, the injury is a lesion caused by asynucleopathic disease.

In various embodiments, the synucleopathic disease is Parkinson'sdisease or dementia with Lewy bodies.

In various embodiments, the injury is a lesion caused by anamyloid-3-mediated disease.

In various embodiments, the amyloid-3 mediated-disease is Alzheimer'sdisease.

In various embodiments, the drug affects blood-brain barrier integrityor function. In various embodiments, the drug is methylene blue,mannitol, bradykinin, or serotonin. In various embodiments, the drugpromotes neuronal health or stability. In various embodiments, the drugis P7C3, a brain-derived neurotrophic factor, a nerve growth factor, acalpain inhibitor, or a flavonoid.

In various embodiments, the drug is localized to a particle and theparticle comprises iron oxide and a magnetic field is applied to thesite of injury, thereby promoting monocyte homing to the site of injury.

In various embodiments, the drug is localized to a particle and theparticle is a capsule and the drug is contained within the capsule andultrasound is applied to the sight of injury, thereby rupturing thecapsule.

In various embodiments, the particle occupies the intracellular space ofthe monocyte.

In various embodiments, the particle attaches to the surface of themonocyte.

In another aspect, the invention provides a method of delivering a drugto the site of an injury in a patient comprising administering to thepatient at least an agent that increases monocyte homing, and aneffective amount of a drug, wherein the agent and the drug enter orattach to at least one monocyte, the endogenous monocyte travels to thesite of injury in the patient; and the drug is released from themonocyte, thereby delivering the effective amount of the drug to thesite of injury.

In various embodiments, the at least one monocyte is an endogenousmonocyte. In various embodiments, the at least one monocyte is anexogenous monocyte. In various embodiments, one or more of the agent andthe drug are localized within one or more particles within the monocyte.In various embodiments, the agent and the drug are both co-localizedwithin one or more particles within the monocyte.

In another aspect, the invention provides a composition comprising atleast one monocyte comprising an effective amount of a drug.

In another aspect, the invention provides a method of delivering a drugto a site of injury of a patient comprising administering to the patienta composition comprising at least one monocyte comprising an effectiveamount of a drug, wherein the drug is released from the intracellularspace of the at least one monocyte at the site of injury, therebydelivering the effective amount of the drug to the site of injury.

In various embodiments, the composition is delivered locally at the siteof injury.

In various embodiments, the composition is delivered systemically.

In various embodiments, the drug is localized within one or moreparticles within the monocyte.

In various embodiments, the particle has a cross-sectional dimensionbetween 1 nm and 50 μm.

In various embodiments, the drug is a drug that affects liver functionor health.

In various embodiments, the particle comprises a cationic polymer.

In various embodiments, the particle comprises a targeting ligand.

In various embodiments, the targeting ligand promotes selective uptakeby an endogenous monocyte.

In various embodiments, the particle comprises a molecule that enhancesendosomal escape.

In various embodiments, the particle comprises a fluorescent dye.

In various embodiments, the fluorescent dye is Nile red or Cy5.

In various embodiments, the particle comprises a hydrophobic polymercore.

In various embodiments, the hydrophobic polymer core comprises apoly(lactic-co-glycolic acid).

In various embodiments, the particle further comprises a coating.

In various embodiments, the particle is a phospholipid vesicle and thephospholipid vesicle comprises one or more concentric lipid layers.

In various embodiments, the drug is released from the intracellularspace by monocyte-to-macrophage differentiation or an injury-specificbiological cue.

In various embodiments, the drug is a prodrug that masks a functionalagent and the functional agent is released at the site of injury.

In various embodiments, the site of injury is in the brain of thepatient.

In various embodiments, the injury is a traumatic brain injury.

In various embodiments, the injury is a lesion caused by asynucleopathic disease.

In various embodiments, the synucleopathic disease is Parkinson'sdisease or dementia with Lewy bodies.

In various embodiments, the injury is a lesion caused by anamyloid-β-mediated disease.

In various embodiments, the amyloid-β mediated-disease is Alzheimer'sdisease.

In various embodiments, the drug affects blood-brain barrier integrityor function.

In various embodiments, the drug is methylene blue, mannitol,bradykinin, or serotonin.

In various embodiments, the drug promotes neuronal health or stability.

In various embodiments, the drug is P7C3, a brain-derived neurotrophicfactor, a nerve growth factor, a calpain inhibitor, or a flavonoid.

In various embodiments, the drug is localized to a particle and theparticle comprises iron oxide and a magnetic field is applied to thesite of injury, thereby promoting monocyte homing to the site of injury.

In various embodiments, the drug is localized to a particle and theparticle is a capsule and the drug is contained within the capsule andfurther comprising the application of ultrasound to the sight of injury,thereby rupturing the capsule.

In various embodiments, the particle occupies the intracellular space ofthe monocyte.

In various embodiments, the particle attaches to the surface of themonocyte.

BRIEF DESCRIPTION OF THE DRAWINGS

For a fuller understanding of the nature and desired objects of thepresent invention, reference is made to the following detaileddescription taken in conjunction with the accompanying drawing figure(s)wherein like reference characters denote corresponding parts throughoutthe several views.

FIG. 1 depicts a schematic summarizing one embodiment of the inventionin which fluorescently-labeled poly(lactic-co-glycolic acid) (PLGA)nanoparticles contain anti-inflammatory dexamethasone (dex) as well asneuroprotective drug (e.g., P7C3) and are intravenously administeredafter TBI. Circulating monocytes quickly phagocytose nanoparticles. Dexacts within the cell and stimulates CD163 expression. CD163-expressingmonocytes home to the brain and transport their P7C3 cargo to the siteof injury. Delayed P7C3 release protects neurons from mitochondrialdysfunction and axonal degeneration; CD163 removes pathological iron;and macrophages promote healthy clearance of cellular debris.

FIG. 2A depicts custom MATLAB® code steps to complete feature-basedsegmentation on confocal images.

FIG. 2B-2D depict intracellular particle number (FIG. 2B), intensity(FIG. 2C), and total drug loading (FIG. 2D) inside macrophages overtime.

FIG. 3 is a chart showing that tetramethylrhodamine (TRITC) exhibitsrhodamine-to-rhodamine quenching. TRITC concentration below 31 ug mL⁻¹is directly proportional to fluorescence. However, TRITC concentrationabove 31 ug mL⁻¹ results in decreased fluorescence.

FIG. 4 shows that loading monocyte-derived macrophages (MDM) withparticles does not interfere with cell spreading over time, acharacteristic of monocyte to macrophage differentiation. Cells thatwere untreated (top graph) spread to a similar extent as particle-loadedcells (bottom graph).

FIGS. 5A-5E depict immunocytochemistry for nuclei (DAPI, A) particles(TRITC, B), the cytoplasmic glucocortiocoid receptor (BuGR2, FIG. 5C),and the overlay of the channels (FIG. 5D). Images were pseudocolored toshow BuGR2, TRITC, and co-localization of the two signals as whitepixels (FIGS. 5E and 5F).

FIG. 6 shows that TRITC signal co-localized with BuGR2 signal at one,three, and seven days after particle administration. Cellular nuclei,human glucocorticoid receptor, TRITC particles, and the cytoplasmicreceptor BuGR are expressed on all particle-loaded macrophages. Whitepixels in the bottom row indicate co-localization between TRITC andBuGR2.

FIG. 7 is a chart showing release of DEX from particles that werefabricated with increasing loading of DEX, ranging from 0% w/w to 56%w/w particles. Release of DEX from particles into 1×PBS showed a largeburst release for particles with high DEX loading. DEX release wasdetectable out until day 9 for most particles.

FIG. 8 depicts release of DEX into the extracellular space from cellscontaining DEX particles in their intracellular space. DEX wasdetectable in the extracellular space for seven days after particleadministration.

FIG. 9 depicts that surface staining of CD163 is increased in cells fivedays after being treated with DEX particles. Data is presented asmean+/−SEM. Data was analyzed with an unpaired t-test with Welsh'scorrection to account for potentially inconsistent variance. Theexperimental group was significantly different from control groupp=0.027.

FIG. 10 is a schematic of the experimental paradigm to test particles'efficacy in directing phenotype in the presence of inflammatory stimuli.Particle-loaded MDM were cultured for seven days in either regular mediaor inflammatory media. After seven days the conditioned media wascollected and analyzed for inflammatory protein production.

FIG. 11 depicts TNF protein production from particle-loaded macrophagesafter seven days in vitro. Increasing DEX loading in intracellularparticles was inversely correlated with inflammatory protein production.

FIG. 12 depicts TNF protein production from MDM collected from fourdifferent human donors after seven days in vitro. Increasing DEX loadingin particles inversely correlated with inflammatory protein productionacross all human donors.

FIG. 13 shows that particle-loaded macrophages preserved their capacityto phagocytose objects including E. coli, polystyrene beads, and myelin.Importantly, pre-treatment of monocytes with DEX particles significantlyincreased myelin uptake in the particle-loaded cells.

FIG. 14 is a bar graph showing that DEX-loaded particles can modulateand maintain phenotype in murine macrophages over time. CD80 surfaceexpression, indicative of inflammation, was downregulated in murinemacrophages five days after treatment with DEX particles.

DEFINITIONS

The instant invention is most clearly understood with reference to thefollowing definitions.

As used herein, the singular form “a,” “an,” and “the” include pluralreferences unless the context clearly dictates otherwise.

Unless specifically stated or obvious from context, as used herein, theterm “about” is understood as within a range of normal tolerance in theart, for example within 2 standard deviations of the mean. “About” canbe understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%,0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear fromcontext, all numerical values provided herein are modified by the termabout.

As used in the specification and claims, the terms “comprises,”“comprising,” “containing,” “having,” and the like can have the meaningascribed to them in U.S. patent law and can mean “includes,”“including,” and the like.

As used herein, the term “monocyte” means cells with the potential todifferentiate into macrophages.

As used herein, the term “particle” means any biologically compatiblestructure, typically a nanoparticle or microparticle that may bephagocytosed by a monocyte and carried along with the monocyte as itmigrates through the body. The term places no restriction on compositionor geometry beyond this functional requirement. Particles may assembledand/or aggregated, e.g., through covalent, physical, and/or ionic bonds.Particles may include but are not limited to hydrophobic or hydrophilic,charged or uncharged polymers, metals such as gold or iron, liposomes,vesicles, and carbon structures such as nanotubes or nanodiamonds.

As used herein, the term “localized” encompasses any type of attachmentto, bonding with, or capture by a particle. For example, an agent ordrug as disclosed herein can be located on an outer surface of aparticle, absorbed within the particle, surrounded by a particle (e.g.,within the center of a microcapsule or nanocapsules), and the like.Different compositions can be co-localized in different locations of aparticle. For example, a homing agent can be located on an outer surfaceof a particle to immediately induce monocyte/macrophage homing to aninjury site, while a drug can be located within the particle in order todelay release for a desired period of time.

As used herein, the term “co-localized” encompasses the localization ofboth an agent and a drug on or within the same particle.

As used herein, the term “drug” refers to any substance that isadministered with the intention of eliciting a therapeutic effect. Thedrug may be but is not limited to a small molecule, peptide, protein,antibody, or nucleic acid.

Unless specifically stated or obvious from context, the term “or,” asused herein, is understood to be inclusive.

Ranges provided herein are understood to be shorthand for all of thevalues within the range. For example, a range of 1 to 50 is understoodto include any number, combination of numbers, or sub-range from thegroup consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 (aswell as fractions thereof unless the context clearly dictatesotherwise).

DETAILED DESCRIPTION OF THE INVENTION

The invention is based, in part, on the discovery that monocytes may beexposed to an agent that increases their propensity to home to a site ofinjury and loaded with a therapeutic payload and which is then carriedto a site of injury by the homing action of the monocyte. Once at thesite of injury, the therapeutic payload can be released from themonocyte and may exert its therapeutic effect. The agent may also actwithin the monocyte (or monocyte-derived macrophage), e.g., withoutbeing released from the monocyte, in order to affect the monocyte'sbehavior.

Compositions

In one aspect, the invention provides a composition containing at leastone monocyte having an intracellular space containing an agent thatincreases monocyte homing to a site of injury, and an effective amountof a drug. Some aspects and embodiments do not include the agent thatincreases monocyte homing to a site of injury. The disclosure below isintended to apply both to embodiments with and without the agent thatincreases monocyte homing to a site of injury.

The agent and the drug can be separate, separately incorporated within aparticle(s), or can be co-localized within the same particle(s). Forexample, monocytes/macrophages can be exposed to both an agent and adrug either inside or outside of the subject to load themonocytes/macrophages with both substances. Loading of the drug and/orthe agent through a particle can be particularly advantageous,especially when the compositions are formed in situ, by delaying releaseof the drug, some of which may cause off-target side effects.

In various embodiments, the number of monocytes can be varied asnecessary to conveniently manufacture the appropriate amount of thecomposition or to deliver greater or lesser amounts or concentration ofthe drug. In various embodiments, the monocytes themselves may be from apatient to whom the composition may ultimately be administered, from adonor, or even from various varieties of stem cells. In variousembodiments, the monocytes may be prepared in vitro and may be treatedto alter monocyte phenotype or behavior. In various embodiments, themajor histocompatibility complex (MHC) type is matched between the donorand patient.

In various embodiments, the particle may occupy the intracellular spaceof the monocyte. The intracellular space refers to the interior of thecell as delineated by the cell membrane. The intracellular spaceincludes compartments within the cell including but not limited toendosomes, lysosomes, and the cytoplasm. In other embodiments, theparticle may attach to the surface of the monocyte such that it iscarried along as the monocyte travels without entering the monocyteitself.

In various embodiments, the agent that increases monocyte homing to asite of injury may be any compound that increases the propensity ofmonocytes to migrate towards a site of injury relative to monocytes thathave not been exposed to the compound. In various embodiments, the agentthat increases monocyte homing may be a chemokine receptor-2 (CCR2)modulator. Without wishing to be bound by theory, modulation of CCR2 mayincrease binding of monocyte chemoattractant protein-1 (CCL1), andincrease the propensity of affected monocytes to migrate toward the siteof injury. In various embodiments, the agent that increases monocytehoming may be dexamethasone.

In various embodiments the particle has a maximum cross-sectionaldimension (e.g., a diameter of a circular cross-section) between 1 nmand 50 μm (e.g., 10 nm to 20 μm, 10 nm to 200 nm, and the like). Thesize of the particle influences uptake and release by monocytes and maybe varied, as appropriate, in order to facilitate, by way ofnon-limiting example, efficient loading or specific controlled-releasekinetics of drug delivery.

In various embodiments, the particle is a lipid or phospholipid vesicle.The phospholipid vesicle may include one or more concentric lipidlayers.

In various embodiments, the particle can be a nanocapsules ormicrocapsule including a shell (e.g., a polymeric shell) surrounding aninner volume. Exemplary nanocapsules and microcapsule are described inU.S. Patent Application Publication Nos. 2009/0028797, 2012/0109045, and2014/0213702.

In various embodiments the particle has a hydrophobic polymer core. Invarious embodiments the hydrophobic polymer is poly(lactic-co-glycolicacid). In various embodiments, the particle comprises a coating. Thecomposition of the particle may be varied as appropriate for thedelivery of various drugs, the specific chemical properties of which,such as, but not limited to, solubility and stability, may make variouscompositions of the particle more or less effective for a given purpose.

In some embodiments, the hydrophobic core or polymeric component isadvantageously used in combination with dex. Dex is a smallcorticosteroid that has a relatively hydrophobic chemical structure,allowing it to be easily adsorbed or conjugated to hydrophobicparticles, such as PLGA. The hydrophobic molecule also has the capacityto diffuse through endosomal phospholipid membranes and intracellularlydown-regulate inflammatory NFκB pathways as well as stimulate theproduction of CD163, the iron-sequestering receptor useful inembodiments in which monocyte-derived macrophages clear iron at the siteof injury.

In various embodiments, the particle includes one or more cationicpolymers. In some embodiments, the cationic polymer may be on thesurface of the particle. Various amounts of cationic polymer incombination with other materials or not, may be used to adjust the zetapotential of the nanoparticles. This provides various advantages,including but not limited to tuning particle uptake and release bymonocytes.

In various embodiments, the particle includes a targeting ligand. Invarious embodiments the targeting ligand promotes selective uptake bymonocytes. In various embodiments, the targeted ligand includes ligandsthat engage mannose or scavenger receptors.

In various embodiments, the particle includes a molecule that enhancesendosomal escape, such as containing charged polymers or having sharp,jagged, and/or pointed shapes.

In various embodiments the particle includes a fluorescent dye. In someembodiments, the particles may be monitored by following thefluorescence of the dye. Any fluorescent dye may be used and multipledyes may be used simultaneously for multiplexing applications that maydepend on the application of multiple embodiments of the inventiontargeting overlap regions of the site of injury. In various embodimentsthe dye may be Nile red, Cy5 or ALEXA FLUOR® 488.

In various embodiments, the drug may be any drug that can be effectivelytransported and released by monocytes. In some embodiments, the drugaffects monocyte or macrophage behavior and may be the same or differentthan the agent that increases monocyte homing to a site of injury. Byway of non-limiting example, the drug may shift the M1/M2 macrophageratio either up or down, promote unique phenotypes of macrophages,recruit additional monocytes to a site of injury. In various embodimentsthe drug may be interleukin-4, interleukin-10, interferon-γ ordexamethasone.

In various embodiments the drug affects liver function or health.

In various embodiments, the drug is a prodrug that masks a functionalagent and the functional agent is released at the site of injury. Askilled person will appreciate that there are a variety of differentprodrug chemistries that may be used to achieve this effect. In someembodiments, monocyte to macrophage or macrophage phenotype changestrigger the unmasking of the active agent from the prodrug.

Methods of Delivering Drugs to a Site of Injury

In another aspect, the invention provides a method of delivering a drugto a site of injury of a patient by administering to the patient acomposition comprising at least one monocyte having an intracellularspace, including at least one particle including an agent that increasesmonocyte homing, and an effective amount of a drug, wherein the at leastone monocyte travels to the site of injury where the drug is releasedfrom the intracellular space, thereby delivering the effective amount ofthe drug to the site of injury. The embodiments of the compositiondescribed above are generally suitable for use in this aspect of theinvention.

Once the monocyte reaches the site of injury the drug is released fromthe intracellular space of the monocyte. In some embodiments, theparticle is released from intracellular space, thereby releasing thedrug. In various embodiments, this may be triggered by the death of thecell or may take place by passive diffusion. In other embodiments, theparticle may dissolve or disintegrate and the drug may exit the cell bypassive diffusion. In yet other embodiments the drug diffuses out of theparticle and then out of the cell. In these embodiments, the particlemay elute into the intracellular space during movement of the cell tothe injury site or may remain within the particle until the particleemerges from the cell.

In various embodiments, the release of the drug from the cell may happenin response to biological cues. In various embodiments, the drug isreleased from the intracellular space by monocyte-to-macrophagedifferentiation. For example, release may be triggered by proteins ormolecules that are upregulated during monocyte-to-macrophagedifferentiation. In various embodiments the drug may be released inresponse to increased levels of reactive oxygen species (ROS).

In various embodiments, the site of injury is in the brain of thepatient. In certain embodiments, the present method presents theadvantage that monocytes can traverse the blood-brain barrier anddeliver a drug to the brain. In various embodiments, the drug affectsblood-brain barrier integrity or function. By way of non-limitingexample, the drug may be methylene blue, mannitol, bradykinin, orserotonin. In various embodiments, the drug promotes neuronal health orstability. By way of non-limiting example, the drug may be P7C3, abrain-derived neurotrophic factor, a nerve growth factor, a calpaininhibitor, or a flavonoid.

The methods of the invention may be applied to target any variety ofbrain injury capable of attracting monocytes or that monocytes may beinduced to travel to. In various embodiments, the injury may be theresult of any type of disease or any form of trauma. In variousembodiments the injury is a traumatic brain injury.

In various embodiments, the injury may be caused by a disease associatedwith the misfolding and aggregation of endogenous proteins. In variousembodiments, the injury may be the result of a disease associated withthe aggregation of α-synuclein, i.e., a synucleopathic disease. Invarious embodiments, the synucleopathic disease is Parkinson's diseaseor dementia with Lewy bodies. In various embodiments, the injury is alesion caused by an amyloid-β-mediated disease. In various embodiments,the amyloid-β mediated-disease is Alzheimer's disease.

In various embodiments, techniques may be used to induce monocyte homingto the site of injury, in addition to or as an alternative to chemicalagents. In various embodiments, the particles may include iron oxides orother paramagnetic materials and a magnetic field may be applied to thebody of the patient, inducing monocytes to travel to or accumulate atthe site of injury or enabling imaging through magnetic resonance.

In various embodiments, techniques may be used to alter or induce drugrelease. In some embodiments, the particle has an interior spacecontaining the drug, i.e., is a capsule. In various embodiments, amechanical or chemical signal ruptures the capsule and releases thedrug, either within the intracellular space of the monocyte or not. Invarious embodiments, ultrasound is applied to the site of injury causingthe capsule to cavitate and rupture.

The monocytes need not be prepared in vitro and endogenous monocytes maybe hijacked and used for drug transport to a site of injury. In anotheraspect, the invention provides a method of delivering a drug to a siteof an injury in a patient by administering to the patient (e.g.,intravenously, parenterally, orally, and the like) particles includingan agent that increases monocyte homing and an effective amount of adrug. The particles enter at least one endogenous monocyte. Theendogenous monocyte travels to the site of injury in the patient and theparticle is released from the monocyte, thereby delivering the effectiveamount of the drug to the site of injury.

EXPERIMENTAL EXAMPLES

The invention is further described in detail by reference to thefollowing experimental examples. These examples are provided forpurposes of illustration only, and are not intended to be limitingunless otherwise specified. Thus, the invention should in no way beconstrued as being limited to the following examples, but rather, shouldbe construed to encompass any and all variations which become evident asa result of the teaching provided herein.

Without further description, it is believed that one of ordinary skillin the art can, using the preceding description and the followingillustrative examples, make and utilize the compounds of the presentinvention and practice the claimed methods. The following workingexamples therefore, specifically point out the preferred embodiments ofthe present invention, and are not to be construed as limiting in anyway the remainder of the disclosure.

Example 1: Particles are Stable in the Intracellular Space ofMacrophages Over Time

In order to test if monocyte-derived macrophages (MDM) can bereprogrammed and used as a therapeutic following traumatic brain injury(TBI), polymeric microparticles that can promote and maintain ananti-inflammatory MDM phenotype were fabricated. After administeringdifferent types of polymeric particles to MDM, it was determined thatpoly(lactic-co-glycolic acid) (PLGA) microparticles loaded with a modeldrug were detectable intracellularly for more than two weeks and slowlyreleased drug to the cell's cytoplasm over that time frame.

To test if intracellular PLGA particles were capable of preserving adrug's bioactivity, PLGA particles loaded with dexamethasone (DEX) werefabricated because DEX is anti-inflammatory, promotes phagocytosis,crosses membranes, and has cytoplasmic receptors. After introducingDEX-loaded particles to MDM, particles were rapidly phagocytosed, storedintracellularly, and released DEX intracellularly to direct MDMbehavior. MDM loaded with particles upregulated the phagocytic receptorsfor one week in vitro relative to untreated MDM, suggesting thatreleased DEX altered MDM behavior. To examine if particles could preventMDM inflammatory polarization even in the presence of pro-inflammatorystimuli, particles were fabricated with increasing amounts of DEX andadministered to MDM. Subsequent treatment with inflammatory stimuliprecluded inflammatory polarization for up to one week in vitro in thecells treated with the highest DEX-loaded particles. Finally, cellstreated with DEX particles upregulated phagocytosis of myelin debris,even in the presence of inflammatory stimuli. This work suggests thatexogenously loading MDM with polymeric particles can generate andmaintain anti-inflammatory MDM behavior even in the presence ofinflammatory stimuli.

This project employs innovative drug delivery techniques to harnessendogenous repair systems to improve recovery after TBI. However,persistent inflammation is a problem in many diseases and disordersincluding multiple sclerosis, stroke, epilepsy, Alzheimer's disease,amyotrophic lateral sclerosis, spinal cord injury, myocardialinfarction, and chronic wounds, among others. Because of the ubiquitousnature of macrophage involvement in driving inflammation, research aimedto elucidate mechanisms of inflammation and simultaneously investigatemethods of redirecting inflammation to promote regeneration could informtherapeutic intervention strategies across a wide spectrum ofneurological and peripheral disorders.

TBI affects a large global population every year, but despite thedevastating consequences of neurological trauma and researchers'efforts, therapeutic interventions to help retain neurological functionand prevent neuronal loss remain elusive. Here, an engineering approachwas utilized to investigate the innate immune system's capacity tomitigate neuroinflammation and rescue mechanically damaged neuronsfollowing TBI. Utilizing this approach, MDM loaded withanti-inflammatory particles can home to the brain, modulateinflammation, and promote clearance of toxic cellular debris. This maylead to a better understanding of the innate immune system's role inneurodegeneration by redirecting potentially neurotoxic behaviors ofMDM. Exploration of this platform may lead to improved nervous systemrepair and a better understanding of neurobiology after trauma.

To test the stability of PLGA particles following phagocytosis by MDM,particles loaded with the model drug tetramethylrhodamine (TRITC) werefabricated. Thereafter, particles were administered to human primary MDMat a concentration of 20 μg of particles per million cells. Cells wereallowed to incubate with particles for four hours to facilitate particleuptake by monocytes. Thereafter, cells were washed to remove anynon-phagocytosed particles and plated in complete media supplementedwith macrophage colony stimulating factor (MCSF). Cells were imaged atregular intervals over 21 days to characterize particle longevity insidecells. Images were acquired with an OLYMPUS® confocal microscope tocapture differential interference contrast channel and the fluorescentTRITC channel. In order to quantify particle longevity inside cells overtime a novel MATLAB® script was generated in order to completefeature-based segmentation image processing (FIG. 2A). In brief, thedifferential interference contrast channel was isolated, converted tobinary, regions of high contrast were identified, and these pixels weredilated. Thereafter, small noise was eliminated, holes inside objectswere filled, and cells on the border of the image and in clusters ofcells were removed to prevent any data skewing. The cell segmentationwas then applied on top of the raw image to validate accurate cellsegmentation. In parallel, the TRITC channel was isolated, converted tobinary, a distance transform was applied in order to segment objectsaccording to a Watershed Transform. The particle segmentation was thenapplied on top of the raw image to validate accurate particlesegmentation (FIG. 2A). Once segmentation for each image was complete,cell size, number of particles per cell, particle intensity, and totaldrug loading per cell was extracted.

An increase in particle number, particle intensity, and total drugloading (defined as the number of particles multiplied by the particleintensity per cell) was observed up until day 3, followed by a decreasein all three metrics thereafter (FIGS. 2B-D). Importantly, data from day1 and day 2 may underrepresent the number of intracellular particles andtheir intensities because TRITC exhibits rhodamine-to-rhodaminequenching, where increasing concentration of TRITC correlates to anincrease in fluorescence up until 31 ug mL-1 (FIG. 3). Thereafter, anysubsequent increase in TRITC concentration correlates with a decrease inTRITC fluorescence (FIG. 3). Because of this quenching behavior, theremay be many particles with high TRITC loading that are not fluorescentlydetectable at early time points. Additionally, it was observed thatphagocytosis of particles did not interfere with monocytedifferentiation into macrophages because cell spreading over time wasconserved in particle-loaded and untreated macrophage conditions (FIG.4).

Example 2: Intracellular Particles Release Model Drugs, which Localizewith Cytoplasmic Receptors

The particle longevity data suggests that the PLGA particles are durableenough to survive intracellularly for over two weeks. However, it isimportant to test if particles were able to release drugs to thecytoplasm of cells. Cytoplasmic delivery of molecules is importantbecause the cytoplasm houses a vast number of molecules that could beregulated by therapeutics. In order to test if a drug could be deliveredto the cytoplasm, PLGA particles loaded with the model drug TRITC wereutilized. Particles were administered to cells for four hours and anynon-phagocytosed particles were removed from the system. After fivedays, the cells were fixed and stained with the cytoplasmic,glucocorticoid receptor BuGR2. Following immunocytochemical staining,the cells were imaged on an OLYMPUS® confocal microscope. Signal fornuclei (DAPI, FIGS. 5A and 5D), particles (TRITC, FIGS. 5B and 5D), andthe cytoplasm marker (BuGR2, FIGS. 5C and 5D) were evident. Toinvestigate if the TRITC signal co-localized with the cytoplasmicsignal, BuGR2 signal was pseudocolored to green, TRITC signal wasexpressed as red, and regions of overlap were expressed as white pixels(FIGS. 5E and 5F). Many white pixels in the zoomed-in, pseudo-coloredimage suggests that TRITC was able to leave the particle, enter thecytoplasm, and co-localize with BuGR2. Additionally, co-localization ofthis stain was important because BuGR2 is a glucocorticoid receptor andthe first therapeutic drug of interest, dexamethasone, is aglucocorticoid.

Repeating the study with multiple time points yielded similar results,where BuGR2 signal and TRITC signal co-localized in the cells (indicatedwith white pixels) over the first seven days following particleadministration (FIG. 6).

EQUIVALENTS

Although preferred embodiments of the invention have been describedusing specific terms, such description is for illustrative purposesonly, and it is to be understood that changes and variations may be madewithout departing from the spirit or scope of the following claims.

INCORPORATION BY REFERENCE

The entire contents of all patents, published patent applications, andother references cited herein are hereby expressly incorporated hereinin their entireties by reference.

1. A composition comprising at least one monocyte comprising: an agentthat increases monocyte homing to a site of injury, and an effectiveamount of a drug.
 2. A method of delivering a drug to a site of injuryof a patient comprising administering to the patient a compositioncomprising at least one monocyte comprising: an agent that increasesmonocyte homing, and an effective amount of a drug, wherein the at leastone monocyte travels to the site of injury where the drug is releasedfrom the intracellular space, thereby delivering the effective amount ofthe drug to the site of injury.
 3. The composition of claim 1, whereinthe agent that increases monocyte homing is a CCR2 modulator.
 4. Thecomposition of claim 1, wherein the agent that increases monocyte homingis dexamethasone.
 5. The composition of claim 1, wherein one or more ofthe agent and the drug are localized within one or more particles withinthe monocyte.
 6. The composition of claim 5, wherein the particle has across-sectional dimension between 1 nm and 50 μm. 7-11. (canceled) 12.The composition of claim 5, wherein the particle comprises a cationicpolymer. 13-17. (canceled)
 18. The composition of claim 5, wherein theparticle comprises a hydrophobic polymer core.
 19. The compositionaccording to claim 18, wherein the hydrophobic polymer core comprises apoly(lactic-co-glycolic acid). 20-37. (canceled)
 38. A method ofdelivering a drug to the site of an injury in a patient comprisingadministering to the patient at least: an agent that increases monocytehoming, and an effective amount of a drug, wherein: the agent and thedrug enter or attach to at least one monocyte, the endogenous monocytetravels to the site of injury in the patient; and the drug is releasedfrom the monocyte, thereby delivering the effective amount of the drugto the site of injury. 39-40. (canceled)
 41. The method of claim 38,wherein one or more of the agent and the drug are localized within oneor more particles within the monocyte.
 42. The method of claim 38,wherein the agent and the drug are both co-localized within one or moreparticles within the monocyte.
 43. A composition comprising at least onemonocyte comprising an effective amount of a drug.
 44. A method ofdelivering a drug to a site of injury of a patient comprisingadministering to the patient a composition comprising at least onemonocyte comprising an effective amount of a drug, wherein the drug isreleased from the intracellular space of the at least one monocyte atthe site of injury, thereby delivering the effective amount of the drugto the site of injury. 45-46. (canceled)
 47. The composition of claim43, wherein the drug is localized within one or more particles withinthe monocyte. 48-55. (canceled)
 56. The composition of claim 47, whereinthe particle comprises a hydrophobic polymer core.
 57. The compositionor method according to claim 56, wherein the hydrophobic polymer corecomprises a poly(lactic-co-glycolic acid). 58-75. (canceled)